[Truncated abstract] G-protein coupled receptors (GPCRs) form one of the largest superfamilies of cell-surface receptors and respond to a vast range of stimuli including light, hormones and neurotransmitters. Although structurally similar, GPCRs are regulated by many diverse proteins, which allow the specific functions of each receptor to be carried out. This thesis focussed on two well-documented GPCRs, the thyrotropin releasing hormone receptor (TRHR) and gonadotrophin-releasing hormone receptor (GnRHR), which control the thyroid and reproductive endocrine pathways respectively. Although each of these anterior pituitary receptors is responsible for distinct physiological responses, both are integral to normal development and homeostasis. This thesis focused on three areas of GPCR regulation: ?-arrestin recruitment, transcription factor regulation and receptor up-regulation. The role of the cytoplasmic protein, ?-arrestin, has perhaps been previously underestimated in GPCR regulation, but it is now increasingly apparent that ?-arrestins not only inhibit further G-protein activation and assist in GPCR internalisation but also act as complex scaffolding platforms to mediate and amplify downstream signalling networks for hours after initial GPCR activation. It is therefore becoming increasingly important to be able to monitor such complexes in live cells over longer time-frames. ... Members of the E2F transcription family have been previously identified by this laboratory as potential GnRHR interacting proteins, via a yeast-2-hybrid screen and BRET. This thesis further investigated the role of E2F family members and demonstrates that a range of GPCRs are able to activate E2F transcriptional activity when stimulated by agonist. However, despite GnRHR displaying robust E2F transcriptional activation upon agonist stimulation, this did not result in any conclusive evidence for functional regulation, although it is possible E2F may modulate and assist in GnRHR trafficking. Furthermore it is apparent that E2F family members are highly redundant, as small effects in GnRHR binding and cell growth were only observed when protein levels of both E2F4 and E2F5 were altered. During the course of the investigation into the effect of E2F transcription on GPCR function, it was evident that long-term agonist stimulation of GnRHR had a profound effect on its expression. As this was explored further, it became clear that this agonist-induced up-regulation was both dose- and time-dependent. Furthermore, altering levels of intracellular calcium and receptor recycling/synthesis could modulate GnRHR up-regulation. In addition, an extremely sensitive CCD camera has been used for the first time to visualise the luciferase activity attributed to GnRHR up-regulation. Overall, this thesis demonstrates the complex nature of GPCR regulation. For the first time, long-term BRET analysis on ?-arrestin interactions with both classes of GPCRs has been examined in a variety of cellular formats. This has given valuable insights into the roles of phosphorylation and internalisation on ?-arrestin interaction. Additionally, this thesis has revealed that prolonged agonist exposure increases receptor expression levels, which has major implications for drug therapy regimes in the treatment of endocrine-related disorders and tumours.

The glycoprotein hormone family comprising of Luteinizing Hormone (LH), Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) plays important role in reproduction and overall physiology of the organism. These hormones are heterodimeric molecules consisting of an identical α subunit non-covalently associated with the hormone-specific β subunit. Both subunits of all these hormones are N-glycosylated. In addition, hCGβ subunit also has four O-linked oligosaccharides located at the C-terminus of the polypeptide(1). The α and β subunits of all these hormones contain five and six disulfide bonds respectively and the crystal structures of hCG and hFSH indicate that both subunits of the hormones belong to the cystine knot family of proteins(2-4). Although the β subunits are hormone specific, there are distinct similarities in these subunits with the 12 cysteines conserved in all these subunits (1). These hormones, because of their unique structural features have proved to be important models for structure–function relationship studies of complex dimeric glycoproteins. Folding of subunits during biosynthesis, role of glycosylation in folding pathways and in vitro and in vivo bioactivity of the hormone, as well as, identification of domains important for subunit association, receptor binding and subsequent signal transduction have been topics of active investigations. The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have unique hormone specific exodomain not present in other members of the GPCR family and characteristic seven transmembrane domains followed by a C terminal domain(5). Primary structure analysis of Glycoprotein hormone receptors family revealed sequence conservation, maximum homology being observed in the transmembrane domain (TMD)(6). Significant homologies could be observed in the hormone specific extracellular domains (ECD) also (7). Despite these homologies, the receptors exhibit exquisite specificity with very low cross reactivity with other members of the hormone family (8). Elucidation of the molecular details of the contacts between the hormone and the receptors has not been achieved so far. Various approaches have been employed to delineate the residues or domains of both hormone and receptors involved in interaction. These include testing of chimeras or mutants of hormones or receptors for changes in activity (9-12), chemical modifications(13) and competition with peptides from either hormones (14) or receptors (15). Polyclonal and monoclonal antibodies against glycoprotein hormones and various fragments of their receptors have been used to determine the role of different domains of both in binding and response (6, 16, 17). However, till date there is no consensus on the specific mechanisms by which the glycoprotein hormone docks onto its receptor. It was proposed that the initial contact between the hormone and the receptor occurs through high affinity binding of the hormone specific β subunit to the Leucine rich regions of the ECD that results in conformational changes in both hormone, as well as, the receptor and brings hormone/ECD complex closer to the TMD of the receptor. The secondary, relatively lower affinity interactions between the hormone and the receptor then take place through common α subunit and exoloops of TMD of the receptor resulting in signal generation (18, 19). Recently a different kind of model has been proposed which suggests that the hormone does not make any direct contacts with the TMD of the receptor. The signal is transduced by the change in contacts between ECD and TMD brought about by hormone’s interaction with ECD(8, 20). The present study was initiated with an overall objective of understanding the molecular details of the hormone receptor interactions of this family, particularly hCG- LH receptor interactions. Two different approaches were employed for this purpose; the first, direct approach being structure elucidation of the members of the glycoprotein hormone family while the second approach uses antibodies against hCG as tools to probe into hormone-receptor interactions. The results obtained using these two approaches have been consolidated in the present thesis and are organized as follows. Chapter 1 is an extensive review of the literature and it builds background for the present work while the exact aim and scope of the present work have been defined in Chapter 2. Chapter 3 describes cloning, expression and purification of recombinant glycoprotein hormones hLH, hCG and single chain derivative of hCG. The Chapter 4 gives details of the molecular aspects of hCG-LH receptor interaction dissected using hCG monoclonal antibodies (MAbs). Chapter 5 discusses implications of the observations made in the present study and states the future directions envisaged. There are a number of endocrinopathies associated with abnormal levels of glycoprotein hormones and treatments of such disorders often demand large quantities of either agonists or antagonists of the hormones. The structure-function relationship studies should help in identifying domains/residues important for subunit interaction, receptor binding, and signal transduction, which would also allow engineering of agonists and antagonists of hormone action. However, structure determination of the glycoprotein hormone family using X-ray crystallography has proved to be a difficult task and it is believed that the heterogeneity in glycosylation is the primary reason for this low success rate in the process of crystallization. The first crystal structure of hCG was that of completely deglycosylated hCG but such a molecule displays antagonistic behavior(2, 3). Use of NMR spectroscopy, the alternate method commonly used for structure determination is often limited by the availability of large quantities of biologically active hormones free of any contaminants. Large quantities of LH, hCG and FSH are also required for treatment of infertile patients suffering from gonadotropin deficiency. The first goal of the present study was thus to produce and purify biologically active recombinant hCG and hLH. Owing to the inherent features of glycoprotein hormones and their potential therapeutic applications, the recombinant expression of these hormones is an important goal from both basic research, as well as, commercial point of view. Considering the above mentioned features it is clear that the expression system used for the hyperexpression of these glycoprotein hormones should also serve as a model system for investigating structure–function relationships and folding of subunits during biosynthesis, in addition to providing sufficient quantities of the hormones for clinical applications. It has been demonstrated that N-linked glycosylation during biosynthesis facilitates protein folding and conformational maturation of glycoprotein hormone subunits into an assembly-competent, biologically active form (21). Therefore, the ideal recombinant expression system should also be able to glycosylate the protein during biosynthesis. The Pichia pastoris yeast expression system was chosen for hyperexpression of glycoprotein hormones as it blends the advantages of both bacterial and mammalian expression systems. Earlier, expression of biologically active hCG and the subunits of hCG and bovine FSH using Pichia pastoris expression system has been reported from the laboratory (22, 23). Chapter 3 (section 3.3.1) of the thesis describes hyperexpression of hLH. The expression of these heterologous proteins was scaled up using fermentation procedures to fulfill the requirements of large quantities of hormones for various applications. Purification of Pichia expressed hormones turned out to be a complex task as large quantity of the hormone was secreted out in the fermentation medium (10litre volume) that was of high ionic strength. Of several different strategies attempted for concentration and partial purification of recombinant hCG, hydrophobic interaction chromatography (HIC) using Phenyl Sepharose matrix emerged as the most efficient technique as a first step of purification. Subsequently, cation exchange chromatography using SP- Sepharose matrix yielded completely purified biologically active recombinant hCG (section 3.3.2). The preliminary data also suggested that Pichia cells express a biologically active form of hCG which appeared to be less glycosylated and of lower molecular weight. Using the same protocol purification of hLH, as well as, single chain derivative of hCG, phCGαβ was achieved (section 3.3.3). These recombinant proteins were characterized extensively using various biochemical, as well as, immunological criteria and were shown to be similar to their natural counterparts with respect to their ability to bind LH receptor and to transduce signal as judged by radioreceptorassays and in vitro bioassays respectively. The hydrophobic interaction chromatography proved an important starting point for purification of all the other members of the glycoprotein hormone family expressed using Pichia pastoris expression system. With the availability of purified, biologically active recombinant hCG in large quantities it was now possible to make attempts towards structure elucidation using NMR spectroscopy. The structure determination of such complex proteins by NMR spectroscopy is made relatively easier by labeling the proteins with magnetically more active, stable isotopes of carbon and nitrogen, 13C and 15N respectively however the cost is often prohibitively high. The Pichia pastoris expression system offers simple means of labeling the proteins as the cells can be grown on simple salts of carbon and nitrogen such as 13C labeled methanol, 15N labeled ammonium chloride or ammonium sulphate. The Chapter 3 also gives a brief account of the preliminary attempts made to label the recombinant hCG with 15N and the structural studies carried out with the carbohydrate moieties of the recombinant hCG using solution NMR spectroscopy. This work was carried out in collaboration with the laboratory of Prof. J.P Kamerling of the University of Utrecht, Netherlands and the efforts are currently underway to elucidate the complete structure of the Pichia expressed hCG. The common feature of receptors and antibodies against the ligand is that both display very specific, high affinity binding towards the ligand. Hence, it is logical to speculate that the antigen binding regions of the antibodies that inhibit hormone binding and/or response, exhibit homology with distinct domains of the receptor. By identifying the epitopes recognized by such antibodies, it should be possible to predict contact points between the hormone and the receptor. In the present study, this hypothesis has been tested using monoclonal antibodies (MAbs) against hCG recognizing different epitopes in the hormone molecule and having different effects on hormone binding and response (Chapter 4). These MAbs were classified as α subunit specific, β subunit specific or heterodimer specific depending on their abilities to bind either subunit in addition to the hormone itself. Interestingly, it was observed that the hCGβ subunit specific MAbs, as well as, heterodimer specific MAbs inhibited hCG receptor binding and hence the response generated by hCG, while the hCGα subunit specific MAbs inhibited only response to the hormone without interfering in binding (Section 4.3.1). To dissect out these interactions further the epitopes recognized by these antibodies on hCG molecule were determined (Section 4.3.2), single chain fragment variable (ScFv) were generated from each of these antibodies and it was shown that these ScFv retain the functionality of the original antibody (Section 4.3.3). Further, the amino acid sequence of each antibody was determined (Section 4.3.4) and finally shown that the antigen binding domains of antibodies show homology to the distinct regions of the LH receptor on sequence alignments between the two using three different programs (4.3.5). The hCGβ subunit specific MAb 52/28' displayed distinct homology with the ECD of LH receptor while the α subunit specific MAb C10 showed regions homologous to TMD of the receptor and the heterodimer specific MAb E12 was found to be similar to the hinge region of the receptor. This clearly indicates that the β subunit of hCG is in close contact with ECD of the receptor while the α subunit makes contacts with the TMD of receptor. The present study thus supports the existing model of hormone receptor interactions, which states that the hormone first binds to the exodomain of the receptor mainly through its β subunit while the integrity of the α subunit is critical for signaling. (24, 25). Also, the observations made in the present study exhibit an interesting possibility of antigen antibody complexes being used as surrogate models for gaining insights into hormone receptor complex. Further, it has been reported that hCG has immunocontraceptive potential(26). Active and passive immunization studies with hCG in primates and humans have demonstrated the possibility of controlling fertility by the antibodies capable of neutralizing hCG. This forms the basis for female contraceptive vaccine that has undergone Phase II clinical trials in India. The MAb E12 characterized in the present study displayed highly specific binding to heterodimeric hCG exclusively without showing any cross reactivity with hLH (Section 4.3.1). The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCGβ absent in hLHβ (Section 4.3.2). The MAb, IgG or its recombinant single chain fragment variable (ScFv), inhibited response to hCG, but not to hLH (4.3.3). Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception. The MAb E12 can also be used for passive immunization in case of emergency contraception. Another potential application of hCG specific antibodies is in homing and the treatment of tumors that secrete hCGβ subunit. The hCGβ subunit specific MAbs used in the present study 52/12 and 52/28' that inhibited hCG receptor binding as well as response generated by hCG can be used in treating such tumors. The functional ScFvs generated from these MAbs in the present study can be made use of on humanization. Thus, the present study has yielded some important molecules for therapeutic applications besides providing a new platform for structure-function relationship studies of the complex glycoprotein hormones.

Ιστορικά, τα ευρήματα των C.B. Huggins (Huggins and Hodges, 1941; Huggins, 1963) και A.V. Schally (Schally et al., 1984) αποτέλεσαν την απαρχή μιας ερευνητικής πορείας με αφετηρία το πεδίο της νευροενδοκρινολογίας και προορισμό εκείνα της γυναικολογίας και της ογκολογίας. Η γνώση αναφορικά με τη φυσιολογία της ενδογενούς ορμόνης (LHRH) και το ρόλο της στην παθοβιοχημεία των ασθενειών (ενδοκρινικών διαταραχών και ορμονο – εξαρτώμενων καρκίνων) και υπό το πρίσμα της πρωτεύουσας ή υποστηρικτικής θεραπευτικής προσέγγισης, κατέστησε τον ιατρικό ευνουχισμό μέσω της δράσης στον υποδοχέα της LHRH – Ι στρατηγική επιλογής για την καταπολέμηση των ενδοκρινικών διαταραχών και των ορμονο – εξαρτώμενων καρκίνων (καρκίνος του μαστού, καρκίνος των ωοθηκών, καρκίνος του ενδομητρίου, καρκίνος του προστάτη). Μια πληθώρα αναλόγων της LHRH έδωσε και δίνει το παρόν σε προκλινικό και κλινικό επίπεδο, με τη συστηματική έρευνα, σύνθεση και ανάπτυξη να αποδίδουν μόρια – αγωνιστές ή – ανταγωνιστές του υποδοχέα της LHRH – Ι, πεπτιδικής (γραμμική ή κυκλική δομή, stapled peptides) ή μη φύσης ως μοναδιαίες οντότητες ή σε σύζευξη με ένα ευρύ φάσμα κυτταροτοξικών μορίων (Αnderes et al., 2003; Keramida et al., 2006; Persson et al., 2009; Kritzer, 2010; Mezo and Manea, 2010). Η παρούσα διδακτορική διατριβή αποσκοπεί στην in vitro και in vivo αξιολόγηση και φαρμακοκινητική μελέτη καινοτόμων αναλόγων της LHRH – κυκλικής και γραμμικής δομής – στη βάση του ορθολογικού μοριακού σχεδιασμού και αποσκοπώντας σε εναλλακτικές προσεγγίσεις για την καταπολέμηση του καρκίνου του προστάτη (και έτερων ενδοκρινικών διαταραχών), συγκριτικά με το leuprolide (εμπορικά διαθέσιμος αγωνιστής του υποδοχέα της LHRH – Ι). Η παρούσα διδακτορική διατριβή θέτει ως υπόθεση εργασίας πως καινοτόμα ανάλογα της LHRH των ήδη υπάρχοντων στην κλινική με βελτιωμένο φαρμακοκινητικό προφίλ δύναται να αποτελέσουν τη βάση βέλτιστων εναλλακτικών θεραπευτικών προσεγγίσεων με ενισχυμένη αποτελεσματικότητα και μειωμένη τοξικότητα, δρώντας in situ ή/ και προσφέροντας νέες δυνατότητες χορήγησης, εστιάζοντας στην οδό ή/ και τη μείωση της συχνότητας αυτής. Η εν λόγω ερευνητική προσέγγιση εστιάζει στον καρκίνο του προστάτη, μια νόσο που χρήζει εναλλακτικής θεραπευτικής στρατηγικής, δεδομένης της χαμηλής αποτελεσματικότητας αυτής (η νόσος προοδευτικά γίνεται ανεξάρτητη των ορμονών και συνεπώς, μεταστατική), αλλά και της χαμηλής ποιότητας ζωής των ασθενών στη βάση του «συμβιβασμού» με τις οδούς χορήγησης που εφαρμόζονται σήμερα στην κλινική (depot formulations). Επιπρόσθετα και κατά την εκπόνηση της εν λόγω ερευνητικής προσέγγισης αναπτύχθηκαν, επικυρώθηκαν και βελτιστοποιήθηκαν καινοτόμες μεθοδολογίες υγρής χρωματογραφίας – φασματομετρίας μάζας (LC – MS/ MS) σε βιολογικά υγρά – ηπατικά μικροσώματα (μύα, επίμυα, ανθρώπου), νεφρικές μεμβράνες μύα, πλάσμα και όρχεις μύα – και έτερα υποστρώματα (κυτταρικά εκχυλίσματα, υδάτινο περιβάλλον ιχθύων Danio rerio), οι οποίες, εν συνεχεία, συζεύχθηκαν με in vitro ή/ και in vivo βιοδοκιμασίες. Τα πειραματικά ευρήματα συγκρίθηκαν με εκείνα των εμπορικά διαθέσιμων αναλόγων της LHRH, με αγωνιστική (leuprolide) ή ανταγωνιστική δράση (antide, cetrorelix). Πιο αναλυτικά και λαμβάνοντας υπόψην το «νοσηρό» φαρμακοκινητικό προφίλ των αναλόγων της LHRH που χρησιμοποιούνται σήμερα στην κλινική, διερευνήθηκε η in vitro (ηπατικά μικροσώματα μύα, επίμυα και ανθρώπου – νεφρικές μεμβράνες μύα) και in vivo (πλάσμα μύα) πεπτιδική σταθερότητα των υπό μελέτη αναλόγων, συγκριτικά με την LHRH και εμπορικά διαθέσιμα ανάλογα αυτής (antide, leuprolide). Τα ευρήματα επέτρεψαν την αξιολόγηση και ταξινόμηση των αναλόγων του ενδιαφέροντος βάσει πεπτιδικής σταθερότητας (δοκιμασία σάρωσης). Ταυτόχρονα, προσδιορίστηκε το μεταβολικό τους προφίλ, αποκαλύπτοντας τους ευάλωτους πεπτιδικούς δεσμούς, καθώς και τις εμπλεκόμενες ενδοπεπτιδάσες (NEP – EC 3.4.24.11, ACE – EC 3.4.15.1). Τα ευρήματα επιβεβαιώθηκαν περαιτέρω με τη χρήση ειδικών ενζυμικών αναστολέων (ενδεικτικά: DL – Thiorphan). Ο νεφρός βρέθηκε να είναι το πρωτεύον μεταβολικό όργανο. Η ανίχνευση και η ημι – ποσοτικοποίηση των μεταβολικών προϊόντων οδήγησε στον προσδιορισμό σχέσεων δομής – δραστικότητας, αποτελώντας τη βάση του ορθολογικού σχεδιασμού καινοτόμων μορίων. Οι απορρέουσες σχέσεις δομής –δραστικότητας υποστηρίζουν πως (i) η μεθυλίωση της υδροξυλομάδας της Tyr5, (ii) η αντικατάσταση της Pro9 από Aze και (iii) η κυκλοποίηση ενισχύουν την πεπτιδική σταθερότητα των αναλόγων του ενδιαφέροντος. Στα πλαίσια μελετών φαρμακοδυναμικής, αναπτύχθηκε καινοτόμος μεθοδολογία LC – MS/ MS για τον ταυτόχρονο ποσοτικό προσδιορισμό της τεστοστερόνης και των υπό μελέτη αναλόγων σε πλάσμα (0, 05 – 100 ng/mL) και όρχεις μύα (2, 0 – 2000 ng/g). Η τεστοστερόνη θεσπίστηκε βιοδείκτης αποτελεσματικότητας (efficacy) και τοξικότητας (toxicity) στη βάση του καίριου ρόλου που διαδραματίζει το εν λόγω στεροειδές στη φυσιολογία του άξονα υποθάλαμος – υπόφυση – γονάδες και την παθοβιοχημεία των ενδοκρινικών διαταραχών και του ορμονο – εξαρτώμενου καρκίνου του προστάτη. Η φαρμακολογική απόκριση (απελευθέρωση τεστοστερόνης) βρέθηκε πως είναι ειδική και λαμβάνει χώρα μέσω του υποδοχέα της LHRH – Ι. Πιο σημαντικά, επετεύχθη ιατρικός ευνουχισμός, βάσει ιστοπαθολογικών ευρημάτων και προσδιορισμού της τεστοστερόνης (πλάσμα και όρχεις μύα), κατόπιν επαναλαμβανόμενης ενδοπεριτοναϊκής χορήγησης του επιλεγμένου καινοτόμου γραμμικού αναλόγου της LHRH, linearGnRH1. Το ίδιο ανάλογο βρέθηκε να έχει κυτταροστατική δράση σε πειράματα κυτταρικού πολλαπλασιασμού (κύτταρα LNCaP και PC3). Παράλληλα και υπό το πρίσμα του ρόλου της LHRH στην παθοβιοχημεία έτερων ορμονο – εξαρτώμενων καρκίνων (καρκίνος του μαστού, καρκίνος των ωοθηκών, καρκίνος του ενδομητρίου), αναπτύχθηκε, επικυρώθηκε και βελτιστοποιήθηκε καινοτόμος μεθοδολογία LC – MS/ MS για τον ποσοτικό προσδιορισμό της 17β – οιστραδιόλης σε βιολογικά υγρά. Τέλος, αναπτύχθηκε καινοτόμος μεθοδολογία LC – MS/MS για τον ποσοτικό προσδιορισμό πεπτιδίων του ενδιαφέροντος στο υδάτινο περιβάλλον των ιχθύων του είδους Danio rerio. Το καινοτόμο γραμμικό ανάλογο της LHRH, linearGnRH1, δεδομένου του φαρμακοκινητικού/ φαρμακοδυναμικού του προφίλ, δρα υποστηρικτικά ως προς την υπόθεση της παρούσας διδακτορικής διατριβής, σύμφωνα με την οποία καινοτόμα ανάλογα των ήδη υπάρχοντων στην κλινική με βελτιωμένο φαρμακοκινητικό προφίλ δύναται να αποτελέσουν τη βάση βέλτιστων εναλλακτικών θεραπευτικών προσεγγίσεων με ενισχυμένη αποτελεσματικότητα και μειωμένη τοξικότητα, δρώντας in situ ή/ και προσφέροντας νέες δυνατότητες χορήγησης, εστιάζοντας στην οδό ή/ και τη μείωση της συχνότητας αυτής. Το linearGnRH1 δύναται να αποτελέσει τη βάση για τον ορθολογικό σχεδιασμό μορίων (stapled peptides, μιμητές), αποσκοπώντας σε εναλλακτικές θεραπευτικές στρατηγικές για την καταπολέμηση του ορμονο – εξαρτώμενου καρκίνου ή/ και των ενδοκρινικών διαταραχών. Πέραν του linearGnRH1, ας σημειωθεί πως κατά την εκπόνηση της παρούσας διδακτορικής διατριβής συγκεκριμένα καινοτόμα κυκλικά ανάλογα της LHRH (cyclicGnRH1, cyclicGnRH2, cyclicGnRHDL1, cyclicGnRHDL2) εμφάνισαν ένα διακριτό φαρμακοκινητικό προφίλ. Το φαρμακοδυναμικό προφίλ των εν λόγω κυκλικών πεπτιδίων απαιτείται να διερευνηθεί περαιτέρω με την εφαρμογή των καινοτόμων μεθοδολογιών LC – MS/MS που αναπτύχθηκαν, επικυρώθηκαν και βελτιστοποιήθηκαν κατά την εκπόνηση της παρούσας διδακτορικής διατριβής. Συνολικά, η καινοτομία της εν λόγω ερευνητικής προσέγγισης έγκειται (i) στην ανάπτυξη, επικύρωση και βελτιστοποίηση ενός πλήθους μεθοδολογιών LC – MS/MS σε σύζευξη με in vitro και in vivo βιοδοκιμασίες, οι οποίες και αποτελούν ένα διακριτό αναλυτικό εργαλείο και (ii) στο προκλινικό φαρμακολογικό μοντέλο που αναπτύχθηκε με την επιλογή του μύα ως ζωικό πρότυπο. Κοινή συνισταμένη, η εν τω βάθει αξιολόγηση της φαρμακοκινητικής/ φαρμακοδυναμικής των πεπτιδίων του ενδιαφέροντος με το ερευνητικό βλέμμα στα πεπτιδικά φάρμακα.. stapled peptides.. μιμητές.. / The highly influential findings of C. B. Huggins (Huggins, 1963) and A. V. Schally (Schally et al., 1984) brought a new era in the research fields of neuroendocrinology, gynecology and oncology. The knowledge acquired regarding the physiology of LHRH and its role in the pathobiochemistry of the disease resulted in the consideration of medical castration via the LHRH receptor as a well established strategy for the treatment of endocrine disorders (e.g. precocious puberty) and hormone – dependent cancers (breast cancer, endometrial cancer, prostate cancer). Numerous LHRH analogues have been synthesized and evaluated both in the clinic and preclinical level. Overall, systematic work has resulted in the synthesis of LHRH receptor agonists and antagonists, either peptides (linear, cyclic, stapled peptides) or small organic molecules as entities or combined with various cytotoxic molecules (Αnderes et al., 2003; Keramida et al., 2006; Persson et al., 2009; Kritzer, 2010; Mezo and Manea, 2010). Although extensive research has been carried out in the field of hormonal therapy, poor pharmacokinetic properties still characterize LHRH peptide analogues. Poor stability of LHRH analogues compromises efficacy, while the need for their subcutaneous administration (depot formulations) aggravates the quality of life for cancer patients. Nowadays, LHRH analogues in the clinic most likely achieve the desired pharmacologic effects by action primarily on the pituitary and to a much lesser extent by direct antiproliferative effects on tumor cells. Herein, we hypothesize that stable analogues of such super – agonists would be advantageous, either by allowing a reduction in dosing frequency or by allowing the use of analogues that act directly on the tumor, with possible additive effects and subsequent enhancements in efficacy. In this context, the pharmacokinetic/ pharmacodynamic profiles of novel LHRH analogues were determined both in vitro and in vivo. Similarly, commercially available LHRH analogues (leuprolide, antide, cetrorelix) served as positive controls. Novel LC – MS/MS based approaches (LC – MS/ MS methodologies coupled to in vitro and in vivo bioassays) were developed, validated and optimized for the evaluation of the peptide analogues in question (linear or cyclic) in various biological fluids and matrices; (i) mouse, rat and human liver microsomes, (ii) mouse kidney membrane preparations, (iii) mouse plasma and tissue (testis), (iv) egg – water (Danio rerio embryos environment). Furthermore, a novel LC – MS/MS based approach was developed, validated and optimized for the simultaneous quantification of testosterone and peptides in question, upon the intraperitoneal administration of peptide analogues in mice. Testosterone served as an efficacy and toxicity biomarker. Peptide metabolism was thoroughly studied by an LC – MS/MS based approach coupled to an in vitro bioassay (mouse kidney membrane preparations), allowing (i) structural elucidation and semi – quantitation of metabolites (and peptides) as a function of time, (ii) determination of the susceptible to proteolysis peptide – bonds, (iii) structure – activity relationships (SARs) and (iv) peptide ranking (screening assay). Taking into account the role of LHRH in gynecology and hormone – dependent cancers of breast, endometrium and ovary, an LC – MS/MS based approach was developed for the quantification of 17β – oestradiol (efficacy and toxicity biomarker) in mouse plasma upon the intraperitoneal administration of peptide analogues in mice. A novel LC – MS/MS based approach was also developed for the quantification of peptides of interest in the aquatic environment of Danio rerio. Employing the aforementioned analytical tools, peptide stability against proteolysis was evaluated both in vitro (mouse kidney membrane preparations) and in vivo (mouse plasma). LHRH and commercially available analogues served as positive controls. The SARs derived suggested that enhanced stability was achieved by (i) methylation on the hydroxyl group of Tyr5, (ii) replacement of Pro9 by Aze and (iii) cyclisation. Hence, new promising chemical entities could be synthesized and developed on the basis of rational drug design. Metabolic profiles were also determined revealing the susceptible to proteolysis peptide bonds. Susceptible peptide bonds and endopeptidases involved were further confirmed in the presence of specific endopeptidase inhibitors. A facile preclinical mouse model was developed in the context of our objectives. Intraperitoneal administration was selected, since (i) it is a mix – mode type of administration with elements of rapid absorption and (ii) oral administration was not practical due to the low bioavailability of the peptides that were tested. The LC – MS/MS based quantification of the selected bioactive peptides and their corresponding metabolites as well as the selective monitoring of biomarkers (e.g. testosterone) in response to drug dose, in plasma and testes, combined with the appropriate preclinical mouse model, represents a distinctive approach. The mouse model described in this paper is particularly valuable, since (i) the human LHRH receptor is homologous to the mouse receptor (Millar, 2004), (ii) information on in vitro and in vivo stability can be obtained with a relatively small amount of peptide (1 – 2 mg), (iii) information on the LHRH receptor agonism (receptor specific in vivo modulation) can be obtained by using testosterone as a marker, (iv) it allows the determination of the dosing regimen required for efficacy based on action on the pituitary, (v) it can become the basis of follow up experiments on genetically modified mouse animal models or other tumour xenografted mouse models (Sharpless and Depinho, 2006; Morgan et al., 2008). The robust sensitive methodology that was developed for the quantification of testosterone in mouse plasma (0.05 – 100 ng/mL) or determination of testosterone in testes (2 – 2000 ng/g) provides an excellent handle on compound efficacy assessment. Testosterone as an efficacy and toxicity biomarker was found to be specific upon peptide binding to the LHRH receptor. Medical castration was achieved upon the repeated dosing of a selected novel linear analogue (linearGnRH1). Measurements in plasma were further supported by statistical significant testosterone values in testis and histopathological findings (atrophy). Moreover the testis weights of the treated animals were significantly lower in comparison to the control group (atrophy induced by dosing), thus making the differences in testosterone testis concentration between control and peptide treated animals even more pronounced. Although the binding affinity of linearGnRH1 on the LHRH receptor was not as high as the binding affinity of leuprolide (~ 15 nM versus <1 nM), the in vivo efficacy between the two analogues was similar (at the tested dose), suggesting that the enhanced stability or bioavailability of linearGnRH1, compensates for binding affinity differences. LinearGnRH1 was also found to be anti – proliferative upon dosing in LNCaP cells (as potent as the superagonist leuprolide). It is possible that linearGnRH1 can play a significant role for the treatment of hormone – dependent cancers, by acting not only at the pituitary level (thus, suppressing the pituitary – testicular axis), but also by exerting an antitumor activity directly on cancer cells, as has been previously shown for other LHRH agonists (Maudsley et al., 2004; Marelli et al., 2006). Except for linearGnRH1, selected cyclic analogues (cyclicGnRH1, cyclicGnRH2, cyclicGnRHDL1, cyclicGnRHDL2) exhibited a distinct pharmacokinetic profile. Their pharmacodynamic profiles should be evaluated further employing the novel LC – MS/MS based approaches developed and validated in this study. Overall, the novelty of the approach described herein consists of (i) the LC – MS/MS methodologies coupled with in vitro and in vivo bioassays developed, validated and employed that provide a distinct analytical tool and (ii) the facile preclinical pharmacological mouse model developed and employed. The approach aims to the pharmacokinetic/pharmacodynamic evaluation of the peptides of interest towards a new generation of peptide drugs.. stapled peptides.. mimetics. Considering the pharmacokinetic/ pharmacodynamic profiles of linearGnRH1, findings on this novel analogue satisfy our hypothesis according to which stable analogues of the LHRH super – agonists used in the clinic would be advantageous, either by allowing a reduction in dosing frequency or by allowing the use of analogues that act directly on the tumor, with possible additive effects and subsequent enhancements in efficacy. LinearGnRH1 can serve as the platform for the rational drug design of new chemical entities (stapled peptides, mimetics) for the treatment of hormone – dependent cancers and/ or endocrine disorders.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Science Author: Marcela Čuchalová Supervisor: doc. MUDr. Josef Herink, DrSc. Title of diploma thesis: The hypothalamic - pituitary - gonadal axis and epilepsy: mutual relationships The content of the diploma thesis is an overview of the anatomy and physiology of the hypothalamic-pituitary-gonadal axis (HHG). Further chapters are devoted to the influence of epilepsy on HHG function, the effect of HHG hormones on epileptic activity itself. The effect of anti-epileptics on HHG functions will also be elucidated. The second part of the diploma thesis deals with separate chapters - catamenial epilepsy and epilepsy during pregnancy. Keywords: antiepileptic drugs, gonadotropin, hypothalamic-pituitary-gonadal axis, prolactin, sex hormones, temporal lobe epilepsy.

A puberdade é um processo biológico complexo do desenvolvimento sexual que tem início no final da infância e se caracteriza pela maturação do eixo hipotálamo-hipófise-gonadal, pelo desenvolvimento dos caracteres sexuais secundários, aceleração do crescimento e finalmente pela capacidade reprodutiva. Nos últimos anos, o peptídeo kisspeptina e seu receptor KISS1R têm sido fortemente envolvidos na regulação da secreção pulsátil do GnRH hipotalâmico e, portanto, com o início da puberdade humana. Mutações nos genes KISS1R e KISS1 foram identificadas em crianças brasileiras com puberdade precoce central (PPC). Estudos em famílias e em irmãs gêmeas estimaram que 50-70% da variação na idade de menarca pode ser hereditária, porém, até pouco tempo atrás, não se tinha conhecimento de variantes genéticas comuns que influenciassem no tempo de puberdade. Recentemente, quatro estudos independentes de associação ampla do genoma estabeleceram que marcadores genéticos próximos ou dentro do gene LIN28B estavam relacionados com a idade da menarca em mulheres normais. Além disso, mutações recessivas no gene lin28 levaram ao desenvolvimento precoce no C. elegans. Camundongos que superexpressam Lin28a apresentaram um retardo no desenvolvimento sexual. Com base nesses achados, investigamos a presença de variantes conhecidas ou novas nos genes KISS1, KISS1R e LIN28B em um grupo de crianças portadoras de PPC idiopática com o intuito de estabelecermos a prevalência dessas mutações na etiologia do desenvolvimento sexual prematuro em humanos. Cento e sete crianças com PPC (101 meninas e 6 meninos) foram selecionados, incluindo casos esporádicos e familiares. A população controle consistiu de 200 indivíduos adultos com história de desenvolvimento puberal normal em idade apropriada. A região promotora e os três exons do gene KISS1, os cinco exons do gene K1SS1R e os quatro exons do gene LIN28B foram amplificados e submetidos à sequenciamento automático. Uma variante em homozigose no gene KISS1, descrita anteriormente por pesquisadores do nosso laboratório, p.H90D, foi identificada em mais três crianças não relacionadas, portadoras de PPC idiopática. Essa variante está localizada no exon 3 do KISS1, levando a substituição de uma histidina por um ácido aspártico na posição 90 da kisspeptina-1 (p.H90D), correspondendo à região amino-terminal da kisspeptina-54 e estava ausente em 200 controles brasileiros. Estudos prévios in vitro com a variante p.H90D não revelaram alterações na capacidade de ligação ou ativação do KISS1R e na resistência a degradação. As mutações ativadoras p.R386P do KISS1R e p.P74S da kisspeptina, previamente descritas em puberdade precoce central, não foram identificadas no estudo atual. Uma nova e rara variante em heterozigose no gene LIN28B, p.H199R, foi identificada em uma menina brasileira com PPC idiopática. Essa variante está localizada no exon 4 do LIN28B, levando a substituição de uma histidina conservada por uma arginina na posição 199 da proteína (p.H199R) e estava ausente em 200 controles brasileiros. O pai da paciente, que apresentou desenvolvimento puberal normal, era portador da mesma variante em heterozigose. Estudos in vitro revelaram que a variante p.H199R não afeta a função de LIN28B na regulação da expressão do miRNA let-7. Outra variante alélica no gene LIN28B foi identificada numa menina com PPC. Essa variante estava localizada no íntron 2 do gene e uma análise computacional demonstrou que ela não altera o sítio de splicing no RNA maduro. Em conclusão, observamos que mutações nos genes KISS1 e KISS1R têm uma baixa prevalência em crianças com puberdade precoce central idiopática. Descrevemos uma nova e rara variante no gene LIN28B (p.H199R) numa menina com puberdade precoce central e os estudos funcionais do LIN28B selvagem ou contendo a variante p.H199R sugeriram que essa variante não está relacionada ao fenótipo de puberdade precoce / Puberty is a complex biological process of sexual development that begins in the late childhood and it is characterized by the maturation of the hipothalamic-pituitary-gonadal axis, secondary sexual characteristics development, growth acceleration and acquisition of the reproductive capacity. Over the last years, the kisspeptin peptide and its receptor KISS1R have been envolved in the regulation of the pulsatile hipothalamic GnRH secretion and consequently with the beginning of the puberty human. Researchers from our laboratory identified mutations in the KISS1R and KISS1 genes in Brazilian children with central precocious puberty (CPP). Studies performed in families and twins estimated that 50%-70% of the variation in the menarce age can be hereditary, however, until last years, we did not have knowledgment of the influence of commun genetic variants in the puberty time. Recently, four independent Genome-Wide Association Studies established that genetic markers near or inside of LIN28B gene were related with the menarce age in normal women. Furthermore, recessive mutations in the LIN28B gene caused a precocious develpment in C. elegans. Interestingly, mouse that overexpress Lin28a exhibited a sexual development delay. Accordingly with these datas investigated the presence of known or new variants in the KISS1, KISS1R and LIN28B genes in a larger cohort of children with CPP to establish the prevalence of these mutations in the etiology of premature sexual development in humans. 107 children with CPP (101 girls and 6 boys) were selected, including sporadic and familial cases. The control population consisted of 200 adults with normal pubertal development. The promoter region and the three exons of KISS1 gene, five exons of KISS1R and four exons of LIN28B were amplified and automatically sequenced. A homozygous variant previously described by researchers from our laboratory in the KISS1 gene, p.H90D, was identified in more 3 no related children with CPP idiophatic. This variant is located in exon 3 of KISS1, resulting in substitution of a histidine to an aspartic acid at position 90 of kisspeptin-1 (p.H90D), in the amino-terminal region of the protein-54 and was absent in 200 Brazilian controls. Previous studies in vitro with the p.H90D variant did not show alterations in the binding or activation capacity and in the resistance to degradation. The activating mutations p.R386P of the KISS1R and p.P74S of the kisspeptin, previously described in central precocious puberty, were not identified in the present study. A new and rare heterozygous variant in the LIN28B gene, p.H199R, was identified in a Brazilian girl with CPP idiophatic. This variant is located in exon 4 of the LIN28B, resulting in substitution of a histidine to an arginine at position 199 of protein (p.H199R) and was absent in 200 Brazilian controls. Her father, which had normal pubertal development, carried the same heterozygous variant. Studies in vitro revealed p.H199R did not affect the function of Lin28B in the regulation of let-7 miRNA expression. Another allelic variant in the LIN28B gene was identified in a girl with CPP. This variant was located in intron 2 of the gene and an in silico analysis showed that it does not change the splicing site in mature RNA. In conclusion, we observed that mutations in the KISS1 and KISS1R genes have a low prevalence in children with idiopathic central precocious puberty. We described a new and rare variant in LIN28B gene (p.H199R) in a girl with central precocious puberty and functional studies of the wild LIN28B or containing p.H199R variant suggested that p.H199R variant of the LIN28B is not related to the precocious puberty phenotype

A kisspeptina, codificada pelo gene KISS1, é um neuropeptídeo crucial na regulação do início da puberdade. A kisspeptina estimula a secreção hipotalâmica do hormônio liberador de gonadotrofinas (GnRH) após se ligar ao seu receptor GPR54. Mutações inativadoras do GPR54 são atualmente consideradas como uma causa rara de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Recentemente, uma mutação ativadora no receptor GPR54 foi implicada na patogênese da puberdade precoce dependente de gonadotrofinas (PPDG). Com base nesses achados, levantamos a hipótese de que alterações no gene KISS1 poderiam contribuir para a patogênese de distúrbios puberais centrais. O objetivo do presente estudo foi investigar a presença de variantes no gene KISS1 em pacientes com PPDG e HHI. Sessenta e sete crianças brasileiras com PPDG (63 meninas e 4 meninos) e 61 pacientes com HHI (40 homens e 21 mulheres) foram selecionados, incluindo casos esporádicos e familiares em ambos os grupos. A população controle consistiu de 200 indivíduos com história de desenvolvimento puberal normal. A região promotora e os 3 exons do gene KISS1 foram amplificados e submetidos a sequenciamento automático. Duas novas variantes no gene KISS1, p.P74S e p.H90D, foram identificadas em duas crianças não relacionadas, portadoras de PPDG idiopática. Ambas as variantes estão localizadas na região amino-terminal da kisspeptina-54 e estavam ausentes em 400 alelos controles. A variante p.P74S foi identificada em heterozigose em um menino que desenvolveu puberdade com um ano de idade. Sua mãe e avó materna, que apresentavam história de desenvolvimento puberal normal, eram portadoras da mesma variante em heterozigose, sugerindo penetrância incompleta e/ou herança sexo-dependente. A variante p.H90D foi identificada em homozigose em uma menina com PPDG, que desenvolveu puberdade aos seis anos de idade. Sua mãe, com história de menarca aos dez anos de idade, era portadora da mesma variante em heterozigose. Células transfectadas estavelmente com GPR54 foram estimuladas com concentrações crescentes de kisspeptina-54 (kp-54) humana selvagem ou contendo as mutações (kp-54 H90D e kp-54 P74S) e o acúmulo de fosfato de inositol (IP) foi medido. Nos estudos in vitro, a kp-54 P74S apresentou uma capacidade de ativação do receptor GPR54 semelhante à kp-54 selvagem. A kp-54 p.H90D mostrou uma ativação da sinalização do receptor significativamente mais potente que a kp-54 selvagem, sugerindo que essa é uma mutação ativadora. No grupo de HHI, uma nova variante (c.588-589insT) foi identificada em heterozigose na região 3 não traduzida do gene KISS1 em um paciente do sexo masculino. O papel dessa variante no fenótipo de HHI permanece indeterminado. Em conclusão, duas mutações no gene KiSS1 foram descritas pela primeira vez em associação com PPDG. / Kisspeptin, encoded by the KISS1 gene, is an important regulator of puberty onset. After binding to its receptor GPR54, kisspeptin stimulates gonadotropin-releasing hormone secretion by the hypothalamic neurons. Inactivating GPR54 mutations are a rare cause of normosmic isolated hypogonadotropic hypogonadism (IHH). Recently, a unique GPR54 activating mutation was implicated in the pathogenesis of gonadotropin dependent precocious puberty (GDPP). Based on these observations, we hypothesized that mutations in the KISS1 gene might be associated with central pubertal disorders. The aim of this study was to investigate KISS1 mutations in idiopathic GDPP and normosmic IHH. Sixty-seven Brazilian children (63 girls and 4 boys) with idiopathic GDPP and 61 patients with normosmic IHH (40 men and 21 women) were selected. Familial and sporadic cases were included in both groups. The control population consisted of 200 individuals who had normal timing of puberty. The promoter region and the 3 exons of the KISS1 gene were amplified and automatically sequenced. Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in two unrelated children with idiopathic GDPP. Both mutations were absent in 400 control alleles and are located in the amino-terminal region of kisspeptin-54. The p.P74S mutation was identified in the heterozygous state in a boy who developed puberty at 1 yr of age. His mother and maternal grandmother, who had normal pubertal development, were also heterozygous for the p.P74S mutation, suggesting incomplete penetrance and/or sex-dependent inheritance. The p.H90D mutation was identified in the homozygous state in a girl with GDPP, who developed puberty at 6 yr of age. Her mother, who had menarche at 10 yr of age, carried the p.H90D mutation in the heterozygous state. CHO cells stably transfected with GPR54 were stimulated with different concentrations of synthetic human wild type or mutant kisspeptin-54 (KP54) and inositol phosphate (IP) accumulation was measured. In vitro studies revealed that the capacity of the p.P74S mutant KP54 to stimulate IP production was similar to the wild type. The p.H90D kisspeptin-54 showed a significantly more potent activation of GPR54 signaling in comparison to the wild type in vitro, suggesting a gain-of-function mutation. In the IHH group, a heterozygous variant in the 3 UTR of the KISS1 gene (c.588-589insT) was identified. The role of this variant in the IHH phenotype remains to be determined. In conclusion, two KiSS1 mutations were described for the first time in association with GDPP.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-12-19T18:38:39Z No. of bitstreams: 2 Tese - Jalsi Tacon Arruda - 2013.pdf: 4216712 bytes, checksum: ec49a9884c1f4b73b53a9b224c7c4aa1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-12-22T11:01:46Z (GMT) No. of bitstreams: 2 Tese - Jalsi Tacon Arruda - 2013.pdf: 4216712 bytes, checksum: ec49a9884c1f4b73b53a9b224c7c4aa1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-12-22T11:01:46Z (GMT). No. of bitstreams: 2 Tese - Jalsi Tacon Arruda - 2013.pdf: 4216712 bytes, checksum: ec49a9884c1f4b73b53a9b224c7c4aa1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-07-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Infertility affects more couples and assisted reproduction techniques offer a possibility of treatment and the chance of having a child. Thus, the first attempt to ovulation induction is critical to the success of the cycle or even for future attempts is successful. Objective: To compare the protocols using GnRH agonist or antagonist for ovarian stimulation in normo-responders undergoing the first cycle of IVF/ICSI. Methods: we conducted a literature review on the history of ovulation induction controlled by medications. From the data available in the database of electronic medical records SISFERT used in the Laboratory of Human Reproduction (LabRep-HC-FM-UFG) a comparative retrospective observational study was conducted with 50 patients divided into two groups according to protocol: GnRH-agonist (leuprolide acetate 1 mg/day short protocol) or GnRHantagonist (Cetrorelix 0.25 mg/day), which received 150 IU/day of rFSH (follitropin alpha) and 250 µg of rhCG (alpha-coriogonadotrofina) in both groups. Results: Statistically significant differences were observed in the days of stimulation with rFSH, total dose of gonadotropin, days of use of GnRH, GnRH dose and total number of follicles (≥ 16 mm) on the day of the group rhCG GnRH agonist. There was no significant difference in other parameters, however, the number of oocytes retrieved was slightly higher in the GnRH agonist, but fertilization rate was higher in the GnRH-antagonist. Pregnancy rates and clinical chemistry were similar in both groups. Conclusions: although no significant differences in the results analyzed, the use of flexible antagonist protocol facilitates the handling and enables the patient using much lower doses of gonadotropins itself as the antagonist, reducing the cost of treatment when compared to the protocol with GnRH agonist. / A infertilidade afeta cada vez mais casais e as técnicas de reprodução assistida oferecem uma possibilidade de tratamento e a chance de ter um filho. Assim, a primeira tentativa de indução da ovulação é fundamental para o sucesso do ciclo ou, até mesmo, para que tentativas futuras sejam bem sucedidas. Objetivo: comparar os protocolos utilizando agonista ou antagonista do GnRH para estimulação ovariana em pacientes normo-respondedoras submetidas ao primeiro ciclo de FIV/ICSI. Métodos: foi realizada uma revisão da literatura sobre a história da indução da ovulação controlada por medicamentos. A partir dos dados disponíveis no banco de prontuários eletrônicos SISFERT utilizado pelo Laboratório de Reprodução Humana (LabRep–HC–FM–UFG), um estudo observacional retrospectivo comparativo foi conduzido com 50 pacientes distribuídas em dois grupos de acordo com o protocolo: GnRH-agonista (acetato de leuprolide 1 mg/dia protocolo curto) ou GnRH-antagonista (cetrorelix 0,25 mg/dia); e que receberam 150 UI/dia de rFSH (alfa-folitropina) e 250 µg de rhCG (alfa-coriogonadotrofina) em ambos os grupos. Resultados: foram observadas diferenças estatisticamente significativas nos dias de estimulação com rFSH, dose total de gonadotrofina, dias de uso do GnRH, dose total de GnRH e o número de folículos (≥ 16 mm) no dia do rhCG no grupo GnRH-agonista. Não houve diferença significativa nos outros parâmetros, no entanto, o número de oócitos recuperados foi ligeiramente maior no grupo GnRH-agonista, mas a taxa de fertilização foi maior no grupo GnRH-antagonista. As taxas de gravidez química e clínica foram similares nos dois grupos. Conclusões: embora não tenha havido diferenças significativas nos resultados analisados, o uso do protocolo flexível com antagonista facilita a manipulação pela paciente usuária e possibilita doses menores tanto de gonadotrofinas quanto do próprio antagonista, reduzindo o custo do tratamento quando comparado ao protocolo com agonista do GnRH

Objetivos: O objetivo deste estudo foi comparar os índices de Pulsatilidade (IP) e Resistência (IR) das artérias uterinas, o volume uterino e a espessura endometrial após o uso do Sistema Intra-uterino de Levonorgestrel (SIU-LNG) ou do agonista do GnRHa (GnRHa) em pacientes portadoras de endometriose pélvica. Pacientes e métodos: Setenta e nove mulheres voluntárias, com idade entre 18 e 40 anos, foram incluídas neste ensaio clínico comparativo, prospectivo, randomizado e controlado. Dezoito foram eliminadas do estudo baseadas nos critérios de exclusão, As 61 pacientes remanescentes foram divididas em dois grupos: 31 pacientes fizeram parte do grupo SIU-LNG (uma foi excluída antes da inserção por apresentar-se grávida) e 30 fizeram parte do grupo GnRHa. Foram submetidasa exame ultra-sonográfico transvaginal bidimensional no dia em que iniciaram o tratamento (inserção do SIU-LNG ou administração de uma ampola de 3,75 mg de GnRHa por via intra-muscular) e seis meses após, avaliando a espessura endometrial, o volume uterino e os IR e IP das artérias uterinas. Resultados: Ambos tratamentos promoveram redução da espessura endometrial (6.08±3.00mm para 2.70±0.98mm e 6.96±3.82mm para 3.23±2.32mm - média ±SD, grupo SIU-LNG e grupo GnRHa, respectivamente). O volume uterino teve redução no grupo usuário do GnRHa (86.67±28.38cm3 para 55.27±25.52cm3) sem alteração significativa nas usuárias do SIU-LNG (75.77±20.88cm3 para 75.97±26.62cm3). Em relação à ascularização uterina, notamos incremento dos IP das artérias uterinas em ambos os grupos (grupo SIU-LNG: artéria uterina direita de 2.38±0.72 para 2.76±0.99 (média ±SD) e artéria uterina esquerda 2.46±0.70 para 2.87±0.96, e grupo GnRHa: artéria uterina direita 2.04±0.59 para 3.12±0.98 eartéria uterina esquerda 2.24±0.59 para 3.15±0.89). Em relação ao IR das artérias uterinas, observamos incremento no grupo GnRHa em ambas artérias e somente na artéria uterina esquerda no grupo SIU-LNG (grupoSIU-LNG - artéria uterina direita de 0.85±0.08 para 0.88±0.07 e artéria uterina esquerda de 0.86±0.07 para 0.89±0.06, e grupo GnRHa: artéria uterina direita de 0.81±0.07 para 0.93±0.09 e artéria uterina esquerda 0.84±0.06 para 0.93±0.09). No entanto, ao compararmos as diferenças, a elevação foi significativamente maior nas usuárias do GnRHa. Conclusões: Ambos GnRHa e SIU-LNG promoveram redução na espessura endometrial e aumento no IP das artérias uterinas. Houve redução do volume uterino nas usuárias do grupo GnRHa, não se alterando no grupo SIU-LNG. Em relação ao IR, houve incremento em ambas as artérias nas usuárias de GnRHa e somente na artéria uterina esquerda nas usuárias do SIU-LNG. / Objectives:The objective of the present study was to compare the uterine arteries pulsatility index (PI) and resistence index (IR), uterine volume and endometrial thickness changes promoted by the use of the levonorgestrel intrauterine device (LNG-IUD) and the gonadotropin-releasing hormone analogue (GnRHa)in patients with endometriosis. Methods: Seventy nine women aged 18 to 40 years were included in this randomized controlled trial. Eighteen was excluded based on the exclusion criteria. The patients were randomly allocated in two groups: 31 women who used the LNG-IUD (since one became pregnant before insertion and wasexcluded) and 30 who used monthly GnRHa injections. They were submitted to a transvaginal two dimensional ultrasound scan on the day the treatment started and 6 months later, for the evaluation of uterine arteries PI, uterine arteries RI, uterine volume and endometrial thickness. Results: The use of LNG-IUD promoted an ndometrial thickness decrease (6.08±3.00mm to 2.7±0.98mm; mean±SD) as does the use of GnRHa (6.96±3.82mm to 3.23±2.32mm). The uterine volume decreased in the GnRHa group (86.67±28.38cm3to 55.27±25.52cm3), but not in the LNG-IUD group (75.77±20.88cm3 to 75.97±26.62cm3). Uterine arteries PI increased in both groups : Uterine arteries PI: LNG-IUD right uterine arterie 2.38 ± 0.72 to 2.76 ± 0.99 and left uterine arterie 2.46 ± 0.70 to 2.87 ± 0.96, and GnRHa right uterine arterie 2.04 ± 0.59 to 3.12 ± 0.98 and left uterine arterie 2.24±0.59 to 3.15 ± 0.89. Uterine arteries RI increased in both arteries in GnRHa and only in the left uterine arterie in the LNG-IUD :Uterine arteries RI : LNG-IUD right uterine arterie 0.85 ± 0..08 to 0.88 ± 0.07 and left uterine arterie 0.86 ± 0.07 to 0.89 ± 0.06, and GnRHa right uterine arterie 0.81 ± 0.07 to 0.93 ± 0.09 and left uterine arterie 0.84 ± 0.06 to 0.93 ± 0.09 . However, the increase was significant higher in the GnRHa group. Conclusions: Both GnRHa and LNG-IUD promoted an endometrial thickness decrease and an increase in the uterine arteries PI. The uterine volume decreased in women who used GnRHa, but not in those who used LNG-IUD.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this study was to evaluate the use of immunological castration as an alternative to surgical castration in the production of beef cattle. In the research were used 48 Aberdeen Angus male bovines, monitored from initial age of six months and initial average weight of 160 kg at weaning. The animals were randomly distributed in the following treatments: surgically castrated at birth; surgically castrated at weaning; immunocastrated Bopriva® with three doses of vaccine, and immunocastrated Bopriva® with four doses of vaccine. The experimental design used was the completely randomized, with 12 replications. Means were classified by F test and compared by Tukey test with α=.05. There was interaction between date of sampling and treatment for serum testosterone levels in the blood plasma, however at slaughter all steers kept only residual levels of testosterone, characterized as castrated. Steers immunocastrated with three applications of Bopriva® had higher daily weight gain and total weight gain in the finishing phase, in relation to surgically castrated at weaning. However, throughout the evaluation stages, the performance was similar between treatments (P>.05). Steers castrated at birth had higher fat thickness to when adjusted 100 kg of cold carcass and lower chilling loss than castrated immunologically with three doses (P<.05). Immunocastration with three doses of Bopriva® provided increment in muscle participation in relation to surgically ones at both ages, and muscle: bone ratio in relation to castrated at weaning. It also reduced the participation of fat in relation to castrated at birth. Total internal organs in percentage of empty body weight, differ between the two immunocastration protocols, with superiority when applied four doses (3.61 vs 3.39 kg). Steers surgically castrated at birth showed superiority in the sum of sum of internal, toilet and kidney fats, for immunocastrated with three doses, regardless of the way it was expressed. The immunological castration proved to be a viable alternative to surgical castration, not changing the main parameters of economic interest and meat quality attributes, and promotes animal welfare, eliminating the surgery. / O objetivo deste estudo foi avaliar a utilização da castração imunológica, como alternativa a castração cirúrgica na produção de bovinos de corte. Na pesquisa foram utilizados 48 bovinos machos, da raça Aberdeen Angus, monitorados a partir de idade inicial de seis meses e peso médio incial de 160 kg, por ocasião do desmame. Os animais foram distribuídos aleatoriamente nos seguintes tratamentos: castrados cirurgicamente ao nascer; castrados cirurgicamente a desmama; imunocastrados com três doses da vacina Bopriva® e imunocastrados com quatro doses da vacina Bopriva®. O delineamento experimental utilizado foi o inteiramente casualizado, com 12 repetições por tratamento. As médias foram classificadas pelo teste F e comparadas pelo teste de Tukey, com α=0,05. Houve interação entre data de amostragem e tratamento sobre os níveis séricos de testosterona no plasma sanguíneo, todavia na ocasião do abate todos os novilhos mantinham apenas níveis residuais de testosterona, caracterizando-se como castrados. Novilhos imunocastrados com três aplicações de Bopriva® apresentaram maior ganho de peso diário e ganho de peso total, na fase de terminação, que castrados cirurgicamente ao desmame. Entretanto em toda a fase de avaliação o desempenho foi similar entre os tratamentos (P>0,05). Novilhos castrados cirurgicamente ao nascimento apresentaram maior espessura de gordura subcutânea, ajustada para 100 kg de carcaça fria e menor quebra ao resfriamento que castrados imunologicamente com três doses (P<0,05). A imunocastração com três doses proporcionou incremento na participação de músculo em relação aos castrados cirurgicamente nas duas idades, e na relação músculo:osso em relação aos castrados ao desmame, mas reduziu a participação de gordura em relação aos castrados ao nascimento. O total de órgãos internos expressos em percentual do peso de corpo vazio diferiu entre os dois protocolos de imunocastração, com superioridade quando aplicou-se quatro doses (3,61 vs 3,39 kg). Novilhos castrados cirurgicamente ao nascimento apresentaram superioridade no somatório das gorduras internas, de descarte e renal, em relação a imunocastrados com três doses, independente da forma como foi expressa. A castração imunológica mostrou-se como uma alternativa viável em relação à castração cirúrgica, não alterando os principais parâmetros de interesse econômico e atributos de qualidade da carne, além de promover o bem-estar animal, eliminando a intervenção cirúrgica.

Abstract The aim of the present study was to evaluate the efficacy of the first trimester screening for Down syndrome (DS) in an unselected low-risk Finnish population. The study involved 4,617 women who attended screening between the 8th and 14th weeks of pregnancy in 1998-2000. They gave a blood sample for the measurement of pregnancy associated plasma protein A (PAPP-A) and free beta human chorionic gonadotrophin (β-hCG). Of these women, 3,178 also had an ultrasound examination for the measurement of fetal nuchal translucency (NT). The risk figure for every screened woman was calculated using a computerized risk figure program. The risk 1 in 250 was used as a cut-off. The subgroup of screen positives comprised 5.8% of the study group. There were 16 DS cases. The combined method (maternal age, NT and the biochemical markers) detected 77% of the affected pregnancies. NT combined with maternal age gave a detection rate of 69%. Serum markers without NT combined with maternal age found 75% of the Down's. In 49 consecutive singleton in-vitro-fertilization pregnancies, the β-hCG value was more often elevated compared to spontaneous pregnancies, increasing the false positive rate. In 67 twin pregnancies, the serum marker levels were approximately double those in singletons. Smoking reduced PAPP-A by 20% making the smokers more likely to get a positive screening result. To determine the impact of the screening on the live born incidence of DS, two historical populations were compared. The first group was screened by second trimester serum samples (β-hCG and AFP) and the second group by first trimester ultrasound examination. When detection rates were at the same level, the second trimester screening reduced the number of live born Down's children more effectively. In conclusion, the first trimester combined method (maternal age, NT, β-hCG and PAPP-A) for Down syndrome screening is efficient in an unselected low risk population. The biochemical screening is not recommended in IVF-pregnancies.